Identification and purification of vitamin K-dependent proteins and peptides with monoclonal antibodies specific for gamma-carboxyglutamyl (Gla) residues

Novel monoclonal antibodies that specifically recognize gamma-carboxyglutam yl (Gla) residues in proteins and peptides have been produced. As demonstra ted by Western blot and time-resolved immunofluorescence assays the antibod ies are pan-specific for most or all of the Gla-containing proteins tested (factors VII, IX, and X, prothrombin, protein C, protein S, growth arrest-s pecific protein 6, bone Gla protein, conantokin G from a cone snail, and fa ctor Xa-like proteins from snake venom). Only the Gla-containing light chai n of the two-chain proteins was bound. Decarboxylation destroyed the epitop e(s) on prothrombin fragment 1, and Ca2+ strongly inhibited binding to prot hrombin. In Western blot, immunofluorescence, and surface plasmon resonance assays the antibodies bound peptides conjugated to bovine serum albumin th at contained either a single Gla or a tandem pair of Gla residues. Binding was maintained when the sequence surrounding the Gla residue(s) was altered . Replacement of Gla with glutamic acid resulted in a complete loss of the epitope. The utility of the antibodies was demonstrated in immunochemical m ethods for detecting Gla-containing proteins and in the immunopurification of a factor Xa-like protein from tiger snake venom. The amino acid sequence s of the Gla domain and portions of the heavy chain of the snake protein we re determined.